How cells process external stimuli into specific, appropriate responses has become one of the major questions in biology. Recent literature suggests that proteins distinct from receptor, G protein, and effector - commonly referred to as accessory proteins - may influence the transfer of signal from receptor to G protein and/or from G protein to effector. This application focuses on one such accessory protein, Activator of G protein Signaling 3 (AGS3), which was identified in a functional screen for receptor-independent activation of G proteins. AGS3 has a modular domain structure, consisting of seven tetratricopeptide repeat (TPR) domains and four G protein regulatory (GPR) domains. The GPR domains bind Gialpha, compete with Gbetagamma for Gialpha binding, and act as a guanine nucleotide dissociation inhibitor (GDI). The role of the TPR domains in AGS3 function is unknown, and little is known about the pattern of endogenous AGS3 expression, the subcellular distribution of endogenous AGS3 and the regulation thereof, the identification of AGS3 binding partners, or the role of AGS3 in signal processing. The experiments proposed in this application involve the characterization of AGS3 expression in the brain, elucidation of the mechanisms of AGS3 regulation, and will define the role of AGS3 in cell signaling and signal processing. This application presents a multifaceted approach to understand the role of AGS3 in G protein signaling.